A Redox-active PARP Inhibitor for Lung Transplantation

描述(由申请人提供)：肺缺血再灌注损伤(IRI)是肺移植(LTX)后的一种急性肺损伤，导致发病率和死亡率。IRI I部分通过氧化和亚硝化物种的合成而介导，这些物种继而导致DNA单链断裂并触发核DNA修复酶聚(ADP-核糖)聚合酶-1(PARP-1)。PARP-1的过度激活导致其底物NAD的消耗，进而耗尽细胞内的能量，导致ATP耗竭和组织坏死。PARP-1的激活也诱导了促炎基因的广泛表达，导致血管通透性、肺水肿、中性粒细胞渗透、肺分流和呼吸衰竭。在IRI损伤模型中，PARP-1的基因缺失或药物抑制有效地减轻了肺损伤，但保护程度是近全的。我们建议通过部署双功能PARP-1抑制剂R-503来更彻底地消除IRI诱导的组织损伤，R-503由两个部分共价连接形成，每个部分都具有组织保护作用：1)PARP-1抑制物部分，2)作为广谱氧化还原降解催化剂的富含硫醇的二氢硫辛基(DHL)结构域。目的#1：在大鼠热缺血肺缺血再灌注损伤模型中，建立双功能PARP-1抑制剂R-503相对于其组分域的优势。大鼠肺原位缺血60min，再灌流4h。缺血前给予静脉注射R-503、DHL、单功能PARP-1抑制剂INO-1001或DHL和INO-1001的联合治疗。通过组织学、PMN浸润水平、脂质过氧化、核因子？B、蛋白亚硝化、PARP-1活化、细胞凋亡和氧合(PaO2)来评估组织损伤。通过检测BAL炎症标志物(蛋白质、中性粒细胞、肿瘤坏死因子和MIP-1)来评估炎症。目的#2：建立同种异体大鼠原位LTX模型，观察R-503的疗效。采用LTX平移模型，供体大鼠在取肺前10分钟静脉注射R-503或赋形剂。冷Perfadex冲洗后，加入R-503(30M)或赋形剂，供体肺在左侧LTX前冷藏12h。右侧供体肺用于免疫组织化学分析PARP-1激活。LTX与左侧供体肺一起LTX后，受者立即接受R-503或赋形剂。R-503的剂量将以AIM#1中阐明的最佳水平为指导。将在3个不同的再灌流时间(1，3，6h)评估受体大鼠的湿/干重比，移植物 氧合、肺血管阻力、动态顺应性、肺F2？-异前列腺素、组织学和免疫组织化学检测3-硝基酪氨酸和聚(ADP-核糖)。具体分析将在3个时间点进行：在再灌注1h，I？B？，磷酸化 分别于再灌注3h、6h、6h测定肺组织细胞密度、蛋白浓度、肿瘤坏死因子-1(MIP-1)、IL-6、IL-1-β的含量。 公共卫生相关性：缺血-再灌注损伤是肺移植后的主要医学并发症，也是导致该人群高死亡率的原因之一。目前还没有批准的预防措施。我们正在开发一种针对这种情况的基本机制的新药，并将在两个临床相关的动物模型中测试这种药物。

DESCRIPTION (provided by applicant): Lung ischemia-reperfusion injury (IRI) is an acute lung injury that contributes to morbidity and mortality following lung transplantation (LTX). IRI i mediated in part by the synthesis of oxidative and nitrosative species that in turn induce DNA single strand breakage and triggering of the nuclear DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP-1). Hyperactivation of PARP-1 results in the consumption of its substrate NAD, which in turn exhausts intracellular energetics, leading to ATP depletion and tissue necrosis. PARP-1 activation also induces a widespread expression of pro-inflammatory genes that contribute to vascular permeability, lung edema, neutrophil infiltration, pulmonary shunt, and respiratory failure. In models of IRI injury, genetic deletion or pharmacologic inhibition of PARP-1 potently reduces lung damage, but the extent of protection is subtotal. We propose a more thorough abrogation of IRI-induced tissue damage via the deployment of a bifunctional PARP-1 inhibitor, R-503, formed from the covalent linkage of 2 moieties, each with demonstrated tissue protection: 1) a PARP-1 inhibitor moiety, and 2) a thiol-rich dihydrolipoyl ("DHL") domain that acts as a broad-spectrum redox degradation catalyst. Aim #1: Establish the superiority of the bifunctional PARP-1 inhibitor, R-503, relative to its component domains, in a rat model of warm-ischemic lung IRI. Rat lungs are rendered ischemic in situ for 60 min and reperfused for 4 h. Prior to ischemia, rats are treated with IV R-503, DHL, the monofunctional PARP-1 inhibitor INO-1001, or a combination of DHL and INO-1001. Tissue damage is assessed by histology, levels of PMN infiltration, lipid peroxidation, NF-?B, protein nitrosation, PARP-1 activation, apoptosis, and oxygenation (PaO2). Inflammation is assessed by examining BAL markers of inflammation (protein, PMNs, TNF-¿, and MIP-1¿). Aim #2: Establish the efficacy of R-503 in a syngeneic rat model of orthotopic LTX. Using a translational model of LTX, donor rats are treated IV with R-503 or vehicle 10 min before lung removal. After flushing with cold Perfadex", spiked with R-503 (30 ¿M) or vehicle, donor lungs are stored cold for 12 h before left LTX. Right donor lungs are processed for immunohistochemical analysis of PARP-1 activation. Immediately following LTX with left donor lungs, recipients will receive R-503 or vehicle. The dose of R-503 will be guided by the optimal level elucidated in Aim #1. Recipient rats will be evaluated at 3 different reperfusion times (1, 3, 6 h) for wet/dry weight ratio, graft oxygenation, pulmonary vascular resistance, dynamic compliance, and lung F2¿-isoprostane, histology, and immunohistochemistry for 3-nitrotyrosine and poly(ADP-ribose). Specific analyses will be carried out at 3 time points: at 1 h post reperfusion for I?B¿, phosphorylation of MAP kinases, nuclear p50; at 3 h post reperfusion for RT-PCR to quantify lung mRNA concentrations of TNF-¿, MIP-1¿, and Bcl-2; and at 6 h post reperfusion for determination of BALF cellularity, protein concentration, TNF-¿, MIP-1¿, IL6, and IL1-¿. PUBLIC HEALTH RELEVANCE: Ischemia-reperfusion injury is a major medical complication following lung transplantation and contributes to the high mortality in this population. At present there are no approved prophylactic measures. We are developing a novel drug that targets the basic mechanisms of this condition and will test this agent in two clinically-relevant animal models.